1. Field of the Invention
The present invention relates to the identification of new muteins of human basic fibroblast growth factor that are unusually potent stimulators of cell division.
2. Description of the Related Art
Fibroblast growth factors (FGFs) are an evolutionarily conserved, large family of mitogenic proteins that stimulate mitosis in mesodermal and neuroectodermal cell lineages (Basilico, C. and Moscatelli, D., Advances in Cancer Research 59:115-165, Eds. Vande Woude, G. F. and Klein, G. (1992)). These proteins also bind heparin and are often referred to in the literature as heparin binding growth factors (HBGFs). Family members share a high degree of nucleic acid and amino acid sequence homology.
Complementary DNA clones encoding basic FGF (bFGF), one member of the FGF family, have been isolated and sequenced. The protein is found to have 89 to 95% amino acid identity among several species, including human, bovine, and rat (Xenopus bFGF is more divergent, sharing 80% homology with human bFGF). This degree of conservation suggests that all regions of the protein may be functionally important. In humans, bFGF is expressed in four forms: (1) an 18-kDa form (155 amino acids) initiated from an AUG codon; (2), (3) and (4) that are 22, 22.5 and 24 kDA, respectively, and all initiated from the CUG codons resulting in N-terminal extensions of varying lengths relative to the 18 kDa form (Florkiewicz, R. Z. and Sommer, A., Proc. Natl. Acad. Sci. (USA) 86:3978-3981 (1989); Pratts, H., et al., Proc. Natl. Acad. Sci. (USA) 86:1836-1840 (1988)). Additionally, while the different forms of bFGF are localized in different compartments of the cell, there is only limited information relating to the functional significance of such subcellular localization. The biological activity of bFGF on cells in trans is effected through signal transduction after cell surface binding to an FGF specific receptor and to heparin sulfate proteoglycans (Moscatelli, D., J. Cell Physiology. 131:123-130 (1987)). In all tissues so far examined, bFGF is found to be expressed, perhaps reflecting its broad spectrum mitogenic activity.
Due to its ability to stimulate the proliferation of a wide variety of cell types, bFGF plays a significant role in many biological processes: (1) angiogenesis (Folkman, J. and Klagsbrum, M., Science 235:442-447 (1987); (2) wound healing (Slavin, J., J Pathology 178: 5-10, 1996; McGee, G. S., et al., J. Surg. Research 45:145-153 (1988); (3) embryogenesis (Kimelman, D., et al., Science 242:1050-1056 (1988); Herbert, J. M., et al., Dev. Biol. 138:454-463 (1990); and (4) tumorigenesis (Ulrich, R., et al., Cancer Cell 3(8):308-311 (1991); Nguyen, M., et al., J. Natl. Cancer. Inst. 86(5):356-361 (1994); Wright, J. A., et al., Crit. Rev. Oncogenesis 4(5):473-492 (1993)). Also there is evidence indicating that bFGF may be used therapeutically in the treatment of cerebral ischemia (Lyons, M. K., et al., Brain Research 558:315-320 (1991); Koketsu, N., etal., Annals Neurology 35(4):451-457 (1994), cerebral aneurysyms (Futami, K., et al., Stroke 26(9):1649-1654 (1995) and neural injury (Logan, A. and Berry, M., TIPS 14:337-343 (1993); Eckenstein, F. P., J. Neurobiology 25(11):1467-1480 (1994); Gomez-Pinilla, F., et al., J. Neuroscience 15(3):2021-2029 (1995)), in addition to its therapeutic potential in the treatment of vascular disease (Richard, J-L., et al., Diabetes Care 18(1):64-69 (1995); Lindner, V., et al., J. Clin. Invest. 85:2004-2008 (1990); Lazarous, D. F., et al., Circulation 91(1):145-153 (1995)) and gastric and duodenal ulcers (Folkman, J., et al., Ann. Surg. 214(4):414-427 (1991); Szabo, S., et al., Scand. J. Gastroenterology 30 Suppl. 208:3-8 (1995); Kitijima, M., et al., Microvasc. Research 50:133-138 (1995); Kusstatscher, S., et al., J. Pharm. Exp. Therapeutics 275:456-461 (1995); Sazbo, S., et al., Gastroenterology 106:1106-1111 (1994); Konturek, S. J., et al., Gut 34:881-887 (1993)).
In order to more fully understand this widespread, biologically significant ligand-receptor system, basic research in this field is focused on elucidating the relationship between bFGF protein structure and function. Early structural studies utilized synthetic peptides corresponding to different regions of the bFGF protein to grossly map the heparin binding and receptor binding regions of the protein (Baird et al., Proc. Natl. Acad. Sci. (USA) 85:2324-2328 (1988); Baird et al., J. Cell Phys. Suppl. 5:101-106 (1987)). More recently, high resolution X-ray crystallography studies (Zhu et al., Science 252:90-93 (1991); Zhang et al., Proc. Natl. Acad. Sci. (USA) 88:3446-3450 (1991); Eriksson et al., Proc. Natl. Acad. Sci. (USA) 88:3441-3445 (1991)) have been used in structure-based, site-directed mutagenesis analyses (Thompson et al., Biochemistry 33(13):3831-3840 (1994); Springer et al., J. Bio. Chem. 269(43):26879-2688 (1994)) and biophysical characterizations of the interactions of bFGF with the bFGF receptor and heparin (Pantoliano et al, Biochemistry 33:10229-10248 (1994)) to further define the functional domains of the protein.
The studies of Thompson et al., Springer et al. and Pantoliano et al. have established the following: (1) the primary receptor binding domain (site 1) is a discontinuous domain, important points of contact being amino acids Y24, R44, N101, Y103, L140 and M142, which are exposed to solvent; (2) a secondary receptor binding domain (site 2) (approximately 250 fold weaker binding) is important for bFGF mitogenicity and comprises amino acids 106-115, which form a type-1 xcex2-turn; and (3) the heparin binding domain which is also a discontinuous domain, the key amino acids of which are K26, N27, R81, K119, R120, T121, Q123, K125, K129, Q134 and K135. (Note: letter/number designations correspond to the single letter amino acid code followed by the position in the linear amino acid sequence for bFGF as described by Zhang et al., 1991.)
One current model of bFGF action suggests that the monomeric ligand bFGF binds to its cell surface receptor through both the high affinity domain (site 1) and the low affinity domain (site 2), leading to receptor dimerization and signal transduction. Heparin binding, known to be important for bFGF activity, is believed to promote site 2 binding to the receptor. (Pantoliano et al., Biochemistry 33:10229-10248 (1994)).
The amino acid sequence of wild-type bFGF is disclosed in several publications: for example, U.S. Pat. Nos. 5,155,214; 4,994,559; 5,439,818; 5,604,293; European Patent Publication No. EP 0 237 966 A2, to name a few.
Structure/function information is useful in studying the biological activity of variants of the bFGF protein sequence. Consequently, there is a great deal of interest in generating new muteins of bFGF for study. For example, analogs in which at least one amino acid is substituted, preferably targeting Cys, Asp, Arg, Gly and Val residues, have been reported (International Publication No. WO 91/09126). Another publication describes a replacement mutein in which at least one cysteine residue is substituted with another amino acid, and deletion mutations in which either 41 amino acids are missing from the amino terminus or 61 amino acids are missing from the carboxyl terminus (U.S. Pat. No. 5,478,740). Mutations in the heparin binding domain of human bFGF are known to alter its biological activity (Heath et al., Biochemistry 30(22):5608-5615 (1991)) and the highly conserved Arg 40 and Arg 45 residues are necessary for stability and mitogenicity of bFGF (Arakawa et al., J. Protein Chemistry 14(5):263-274 (1995)). Additionally, enhanced stability analogs have been reported in which 2 or 3 amino acids are added, deleted or substituted, with serine substitution being preferred for cysteine residues (Eur. Pat. Pub. No. EP 0 281, 822 B2).
As previously disclosed, bFGF is a powerful mitogen and a key regulatory factor in many biological processes: for example angiogenesis, wound healing, ischemic tissue repair, gastric and duodenal ulcer healing, tumorigenesis and neural tissue survival and regeneration. Not surprisingly, the therapeutic value of wild-type and mutein bFGF""s is recognized and detailed in the art, some examples of which are listed herein. Therapeutic treatments related to the above disclosed biological processes have been described for wild-type bFGF in U.S. Pat. Nos. 5,612,211; 5,439,818; 5,604,293; 5,514,566; 4,994,559; 5,514,662 and European Patent Application No. EP 0237966 A2. Similar therapeutic treatments utilizing bFGF muteins are disclosed in U.S. Pat. Nos.5,132,408; 5,352,589; 5,360,896; 5,371,206; 5,302,702; 5,310,883; 5,478,804; 5,576,288 and European Patent Application No. EP 0 281 822 A2. For example, replacement muteins in which the loop region of human bFGF (amino acid residues 118-122) are replaced with selected peptides of other FGF family members are described in U.S. Pat. No. 5,491,220. These muteins are disclosed to be useful in the treatment of cancer as antiproliferative agents or as agents that promote vascularization.
Given the potential therapeutic value of the bFGF protein, there is a need in the art for the development of novel analogs of bFGF with improved biological properties.
It is therefore an object of the present invention to provide analogs of human bFGF with superagonist activity. Other objects, features and advantages of the present invention will be set forth in the detailed description of the preferred embodiments that follows, and in part, will be apparent from the description or may be learned by practice of the invention.
In a first embodiment, the present invention is directed to muteins of human bFGF in which Glutamate 89 or Aspartate 101 or Leucine 137 or combinations or permutations thereof are substituted with a neutral and/or hydrophobic amino acid. Other embodiments are drawn to polynucleotides encoding the muteins of the first embodiment, a vector containing said polynucleotide and a host cell carrying said vector. A third group of embodiments are drawn to processes to produce a polypeptide, to produce cells capable of producing said peptide and to produce a vector containing DNA or RNA encoding said polypeptide. A fourth group of embodiments are drawn to methods to stimulate cell division, to heal a wound, to treat ischemia, to treat peripheral vascular disease, to treat a gastric or duodenal ulcer, to treat neural injury and a pharmacologic composition comprising an effective amount of the mutein of the first embodiment.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.